Does Gibberellic Acid Induce the Transfer of Lipase from ... - NCBI

Plant Physiol. (1987) 85, 487-496 0032-0889/87/85/0487/10/$01.00/0

Does Gibberellic Acid Induce the Transfer of Lipase from Protein Bodies to Lipid Bodies in Barley Aleurone Cells?' Received for publication May 19, 1987 and in revised form June 26, 1987

DONNA E. FERNANDEZ*2 AND L. ANDREW STAEHELIN

Department ofMolecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347 ABSTRACT We have examined the effect of gibberellic acid (GA3) on the distribution of the enzyme responsible for mobilizig storage triacyglycerol in aleurone cells of HordeEm Pulgare L. cv Himalaya. Using cellular fractionation techniques, we find that, in cells that have not been exposed to hormone, neutral lipase activity is principally associated with a pellet containing the membranes of protein bodies. If the cells are exposed to GA3 for at least 1 hour, the majority of the lipase activity becomes associated with the lipid body fraction. The nature of the in vivo association between lipid bodies and protein bodies was examined using ultrarapid freezing followed by freeze-fracture electron microscopy. Our analysis indicates that the phospholipid monolayer surrounding the lipid body is directly continuous with the outer leaflet of the bilayer surrounding the protein body. Based on our data, we propose that lipase can be transferred from protein bodies (storage form) to lipid bodies (active form) by lateral diffusion within the plane of the fused phospholipid monolayer, and that the transfer can be controlled by gibberellic acid by an unknown mechanism.

When barley aleurone cells are exposed to the hormone giberellic acid (GA3), the cells are dramatically restructured in terms of gene expression and the organization of subcellular structures. Within 4 to 8 h, increased synthesis of a-amylase (17) and proliferation of rough ER (22) become apparent. Since both phospholipid precursors and the energy needed to support increased synthetic activity are principally derived from triacylglycerols stored in lipid bodies in aleurone cells (8), it seems likely that changes in lipid metabolism may also occur quite rapidly after the cells are exposed to GA3. Previous studies indicate that changes in lipid metabolism are among the earliest effects caused by GA3 in barley aleurone cells. Within 2 h after the cells are exposed to GA3, the in vitro activities of two enzymes involved in phospholipid synthesis, phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholineglyceride transferase (EC 2.7.8.2), increase (1, 21). Within 4 h, increased incorporation of radiolabeled choline into a microsomal fraction (10) and of radiolabeled orthophosphate into phospholipids (23) is observed. On the other hand, radiolabeled acetate and glycerol are not incorporated into phospholipids to any great extent in aleurone cells (14, 23, 32, 35). Instead, it appears that precursors derived from the preexisting storage lipids

are predominantly used for new phospholipid synthesis (35). Few investigators have looked at the processes that supply the phospholipid precursors and energy in aleurone layers, i.e. the reactions involved in the hydrolysis of triacylglycerols. Enzymes involved in the release of energy from fatty acids via the glyoxylate cycle appear within 1 to 2 d of germination in whole wheat seeds and can be induced by GA3 in embryo-less seeds (9). Lipases that are involved in triacylglycerol hydrolysis also appear in whole wheat seeds after 1 d of germination but cannot be induced by GA3 in embryo-less seeds (30). Jelsema et al. (20) have shown, however, that isolated wheat aleurone layers that have not been exposed to GA3 do contain acid lipase activity. In addition, their observations present an interesting enigma. Although triacylglycerols, the apparent substrate, are found within lipid bodies, the acid lipase activity is located principally in a pellet containing protein bodies. When the cells are exposed to

GA3, the lipase activity disappears from the pellet (20). We have reexamined the distribution of lipase and GA3induced changes in that distribution more closely using cellular fractionation techniques and electron microscopy. Our biochemical data indicate that lipase is associated with protein bodies as well as with lipid bodies. The distribution of lipase between these two organelles appears to be controlled by GA3. Since our structural data indicate that lipid bodies can bind to the surface of protein bodies and that the monolayer that surrounds the lipid body becomes continuous with the outer leaflet of the bilayer that surrounds the protein body, we propose that lipase may be transferred between the two organelles by lateral diffusion. A preliminary report of our biochemical findings appeared in Fernandez and Staehelin (13).

MATERIALS AND METHODS Plant Materials. Aleurone layers of Hordeum vulgare L. cv Himalaya (1979 harvest, Washington State University, Pullman, WA) were prepared from surface-sterilized, embryo-less half seeds (7) imbibed for 4 d in 50 mm L-arginine (18). Layers were incubated on a rotary shaker at 26°C in 2 mm sodium-acetate buffer (pH 4.8) containing 20 mM CaCl2, 7.5 mg/ml chloramphenicol, and either 10 uM GA3 (Sigma Chemical Co.) or H20. Isolation of lipid bodies. Lipid bodies were isolated using a modification of a procedure developed originally for corn scutella (25). The aleurone layers were homogenized at 4°C using a Virtis 45 homogenizer (Virtis Co., Gardiner, NY) operated at low speed. The homogenization (grinding) medium (10 ml/100 layers) contained 0.6 M sucrose, 1 mM EDTA, 10 mm KCl, 1 mM MgCl2, 2 mm dithiothreitol, and 0.15 M Tricine buffer adjusted to pH 7.5 with KOH. (In most experiments, 5 mm e-aminoca' Supported by National Institutes of Health grant GM 18639 to L. A. proic acid, 1 mM benzamidine-HCl, and 10 mm leupeptin were also added to inhibit proteases.) The homogenate was filtered S. and a National Science Foundation Graduate Fellowship to D. E. F. 2 Present address: Department of Biology, Indiana University, Bloom- through one layer of nylon mesh cloth with a pore size of 30 ,m. The residue retained on the cloth was then ground with sand in ington, IN 47405. 487

488

FERNANDEZ AND STAEHELIN

a small volume of grinding medium, and refiltered. The residue from the second grinding operation was squeezed to obtain the maximum amount of homogenate. Lipid bodies were isolated from the filtrate using a series of sucrose step-gradients (Fig. 1). The filtrate was added to a centrifuge tube and overlaid with an equal volume offlotation medium (same composition as the grinding medium except that the sucrose concentration is lowered to 0.5 M). The step-gradient was centrifuged at 10,000g for 30 min at 4°C in a swinging bucket rotor. The lipid bodies (LB 1) migrate to the surface of the medium and can be collected with a spatula. The pellet was resuspended in a small volume of grinding medium and stored at -20°C. The supernatant fraction (located between the floating lipid bodies and the pellet) did not exhibit lipase activity and was discarded in most experiments. For the experiments in Tables I and II, the collected lipid bodies were resuspended in 2 M NaCl, 0.4 M sucrose, and 0.15 M Tricine-NaOH buffer (pH 7.5) (salt medium I), overlaid with salt medium II (same composition as salt medium I except that the sucrose concentration is lowered to 0.3 M) and recentrifuged at 10,000g for 30 min; there was no observable pellet at this step. The lipid bodies (LB2) were collected and washed twice in stepgradients of grinding and flotation media. The final lipid body pellicle (LB4) was resuspended in a small volume of flotation medium. Fractions were stored at -20°C. For the experiments on lipase distribution (Tables III, IV, and V), the lipid bodies (LB 1) and pellet isolated from the first centrifugation step were resuspended in grinding medium, overlaid with flotation media, and recentrifuged at 10,000g for 30 min. The pellet and lipid bodies from this step were collected in grinding medium (pellet) or flotation medium (lipid bodies) and stored at -20°C. Assays. Neutral lipase activity was measured by mixing the subcellular fractions with a defined triacylglycerol substrate (trilinolein) at pH 7.5 and quantitating the release of free fatty acids B

:,A.'

(25). The substrate was prepared by emulsifying 2.5 mM trilinolein (18:2, Sigma Chemical Co.) for 1 min at room temperature with a probe sonicator (Sonifier cell disruptor, Heat SystemsUltrasonics, Inc., Plainview, NY) in 5% gum acacia, 0.1 M TrisHCI, and 5 mm DTT (pH 7.5). Subcellular fractions were incubated with the substrate in a shaking water bath at 34°C and duplicate 50 X samples were removed every 30 min for 2 h. The samples were extracted once with 3 ml of chloroform:heptane:methanol (4:3:2, v/v) and free fatty acids were detected in the extracted samples using the colorimetric method of Nixon and Chan (27) as modified by Lin and Huang (25). Palmitic acid (16:0) (Sigma Chemical Co.) was used to generate a free fatty acid standard curve. The lipase specific activities in Tables I and II were calculated after measuring the protein content of the subcellular fractions using the Coomassie dye binding assay of Bradford (2) and bovine serum albumin as a standard. Data Analysis. We have chosen to report the data as proportions in order to compare values from different experiments with different total activities. Because lipase activity could not be detected in the supernatant fraction, total activity for a given sample was calculated by adding together the activity in the pellet and lipid body fractions. The activity of each fraction was then expressed as a proportion of this total. Values of 0 and 100% were converted to [l/4n]% and (1-[1/4n])% and the data was arcsine transformed to meet the assumptions of parametric statistical analysis. The data in Table III are reported in terms of 95% confidence intervals which were calculated using transformed data and Student's t distribution for small sample sizes. Electron Microscopy. Monolayers of aleurone cells were prepared as described (12). Subcellular fractions were isolated on sucrose step gradients as described above. Cells and subcellular fractions were ultrarapidly frozen with a propane-jet freezer and processed for freeze-fracture electron microscopy as described (12). Membrane fracture faces were labeled according to Branton et al. (3): the P (protoplasmic) face is the fracture face of the membrane leaflet closest to the cytoplasm; the E (exoplasmic) face is the fracture face of the leaflet closest to the extracellular or endoplasmic space.

filtrate A

= grinding

medium

B

=flotation

medium

C

= salt

medium I

D

=salt

medium

0,000

LB3

x g,

I

30 min

I

RESULTS Isolation of Lipid Bodies. Because of their low density, lipid bodies can be easily isolated from a homogenate of barley aleurone layers using a series of sucrose step gradients and salt washes (25, Fig. 1). Analysis of the various fractions by freeze-fracture electron microscopy indicates that the separation of lipid bodies from other cellular components is very efficient. Lipid bodies and protein bodies can be easily distinguished in freeze-fracture electron micrographs because the lipid bodies are invariably small and round and their contents do not etch, whereas the protein bodies are larger, have etchable contents, and their membranes have a very high density of P-face particles (cf. Fig. 5). The majority of the lipid bodies float up, whereas protein body membranes are found in the l0,OOOg pellet, often as large sheets with some attached lipid bodies. The material isolated from the surface of the sucrose gradient after two centrifugation steps (LB2) consists almost exclusively of lipid bodies (Fig. 2), although small, attached fragments of bilayer membranes are seen occasionally (Fig.

LB41

FIG. 1. Diagram summarizing the sequence of sucrose step gradients used for isolating lipid bodies (LB) from a homogenate of barley aleurone

layers.

Plant Physiol. Vol. 85, 1987

2, arrowhead).

Intracellular Distribution of Lipase Activity. Lipid body fractions isolated from aleurone layers that have not been exposed to GA3 contain measurable amounts of neutral lipase activity. The enzyme in these fractions cleaves free fatty acids from trilinolein, a triacylglycerol containing three 18:2 fatty acids. Since 18:2 fatty acids are also the most abundant fatty acids in cereal acyl lipids (26), the in vivo substrate of this lipase is probably very similar to trilinolein. When the lipid body fraction

LIPASE TRANSFER IN ALEURONE CELLS

489

FIG. 2. Freeze-fracture electron micrograph of lipid bodies (LB2) isolated from untreated cells (4 h). The lipid body (LB) fraction is essentially devoid of other cellular components. Occasionally, small fragments of bilayer membranes (arrowhead) are seen attached to the lipid bodies. LBM, inner surface of lipid body monolayer (x75,000, bar = 0.2 um).

Table I. Co-purification of Lipase Activity and Lipid Bodies Lipid bodies were sequentially purified using a series of sucrose step gradients. Fraction Specific Activity nmol/min mg protein 18 LBI 62 LB2 370 LB4 -

Table II. Effect of GA3 on the Specific Activity of Lipase Associated

with Lipid Bodies Lipid bodies (LB2) were isolated-from barley aleurone layers that had been incubated with or without GA3 for various lengths of time. Specific Activity +GA3/-GA3 Incubation Time -GA3 +GA3 h 1 4 12

mg pronmol/min. tein

104 132 62

72 62 53

ratio 1.4 2.1 1.2

(LB 1) is washed with a high-salt buffer to remove electrostatically bound proteins and then washed several more times in stepgradients of grinding and flotation media (Fig. 1), the lipase activity co-purifies with the lipid bodies and the specific activity of the lipase increases 20-fold (Table I). The amount of lipase associated with the lipid body fraction appears to be controlled by GA3. The specific activity of the lipase in isolated lipid bodies (LB2) increases as much as 2-fold when aleurone cells are incubated with 10 Mm GA3 for various lengths of time before they are homogenized (Table II). The total amount of measurable lipase activity (generally around 20-50 nmol free fatty acid released/min- 100 aleurone layers) can vary from preparation to preparation depending on isolation conditions and the degree of homogenization of the tissue. However, the proportion of total activity that is associated

with a given fraction remains relatively constant (Table III). In uninduced cells, the majority of the lipase activity is associated with the 10,000g pellet. When the cells are exposed to GA3, the proportion that is associated with the pellet decreases and the proportion that is associated with the lipid body fraction increases significantly (Table III). This increase in activity cannot be attributed to more efficient lipid body isolation because the specific activity increases as well (Table II) and, in general, the yield of lipid bodies from GA3-induced layers is lower than the yield from the same number of uninduced layers. The specific activity of lipase in the pellet also decreases when the cells are exposed to GA3 (data not shown). In many experiments, there was no measurable lipase activity in pellets isolated from induced cells (c.f Table IV). Many GA3-induced changes in barley aleurone cells can be blocked by abscisic acid (ABA), including the synthesis of aamylase (6), the formation of polysomes (1 1), and the stimulation of phospholipid synthesis (10, 21, 23). Because ABA is such an Table III. Effect of GA3 on the Distribution of Lipase Activity Total Lipase Activity Treatment Lipid Pellet bodies 2h % 14 + 7 85 ± 7 -GA3 (n = 6) 26 ± 4 73 ± 4 +GA3 (n = 7) Table IV. Effect ofABA on the GA3-induced Shift in Lipase Distribution Total Lipase Activity Treatment Lipid Pellet bodies 2h % 100 0 +GA3(n=l) +ABA 100 0 69 31 +GA3/+ABA

4904FERNANDEZ AND STAEHELIN effective and consistent antagonist of GA3-induced events, its

effect on the shift in total lipase activity from the l0,OOOg pellet to the lipid body fraction was tested. As shown in Table IV, the relative increase in lipase activity in lipid body fractions is also sensitive to ABA treatment. When aleurone layers are exposed to a combination of 1 iM GA3 and 10 pM ABA ([*] cis-trans isomer), the GA3-induced increase in lipase activity is at least partially blocked. ABA alone has no effect on the lipase distribution in the cells. The shift in lipase distribution appears to occur relatively rapidly once aleurone cells have been exposed to GA3. If aleurone layers are exposed to GA3 for 45 min or less, immediately placed on ice and then homogenized, most or all of the detectable lipase activity is found in the l0,OOOg pellet. However, if the layers are exposed to GA3 for 60 min, lipase activity can be detected in the lipid body fraction in some samples. After 2 h of exposure to GA3, the majority of the lipase activity is consistently found in the lipid body fraction (Table V; cf Table III). Freeze-Fracture of Lipid Bodies. It is difficult to interpret results obtained from cellular fractionation studies unless the situation that exists in intact cells is known. Therefore, the spatial organization of lipid bodies in ultrarapidly frozen barley aleurone cells was examined using freeze-fracture electron microscopy. Lipid bodies consist of a core of triacylglycerols surrounded by a phospholipid monolayer (37). When such structures are freezefractured, the fracture planes generally travel straight across the hydrophobic lipid core, exposing a smooth surface which can be easily distinguished from the surrounding cytoplasm (Fig. 3). No particles are present within the hydrophobic core: protein complexes appear to be confined to the margins of the lipid bodies, i.e. to the limiting monolayer. Occasionally, fracture planes will travel between the monolayer and the underlying lipid. Although particles can be seen on the P-faces of such fractures (i.e. the inner surface of the monolayer, Figs. 2, 3, 6), the E-faces are smooth and appear to consist exclusively of lipid (Fig. 3). Lipid Body-ER Associations in Intact Cells. Lipid bodies are often seen near the ER in both GA3-treated and untreated cells (12). Many of these lipid bodies appear to be free in the cytoplasm but, occasionally, direct connections between the lipid bodies and the ER can be observed. The nature ofthis type ofassociation becomes clear when a fracture plane passes through the narrow neck (approximately 30 nm in diameter) that connects the two organelles (Fig. 4). The membranes of the two organelles are in direct contact and, in fact, the lipid body monolayer is continuous with the outer leaflet of the membrane bilayer of the ER. Lipid Body-Protein Body Associations in Intact Cells. Lipid bodies also associate with protein body membranes in both GA3treated and untreated cells. We find, as did Buttrose (4), that the E and P fracture faces of protein bodies in aleurone cells are covered with distinctive 'plaques' (Figs. 5a, 6a). The plaques mark sites where the lipid bodies, which completely surround Table V. Time Course of the GA3-induced Shift in Lipase Distribution Incubation Time

(+GA3)

Total Lipase Activity

240

bodies

%

min

0(n=l) 15 30 45 60 60 120 180

Lipid

Pellet 100 100 100 100 100 36 22 22 24

0 0 0

0 0 64

78 78 76


FIG. 3. Freeze-fracture electron micrograph of lipid bodies within the cytoplasm of a barley aleurone cell (untreated, 7-8 h). Fracture planes generally travel straight across the hydrophobic triacylglycerol core (TG), exposing a smooth surface which can be readily distinguished from the surrounding cytoplasm. No particles are present within the hydrophobic core: protein complexes appear to be confined to the limiting monolayer (arrows). Occasionally, fracture planes will travel between the monolayer and the underlying lipid. Many particles are visible on the inner surface of the lipid body monolayer (LB-PF); however, the other faces of such fractures (*) are smooth and appear to consist exclusively of lipid (x85,000, bar = 0.2 ,m).

the protein bodies, are attached to the protein body membranes. Careful examination of the junction between the two organelles (Figs. Sb; 6, b and c) shows that no half-bilayer leaflet is crossed when the fracture plane passes from the smooth interior of the lipid body to the particle-rich outer leaflet of the protein body

membrane (explanatory diagram, Fig. 7). Thus, the relationship between the lipid bodies and protein bodies is identical to that between the lipid bodies and the ER, i.e. the monolayer of the lipid body is continuous with the outer leaflet of the membrane bilayer of the protein body. As with the ER, the connection between the two organelles consists of a narrow neck-like structure filled with triacylglycerol. The 'necks' are approximately the same diameter as those seen with the ER because pits of approximately 30 nm in diameter can be seen on the P-face of protein bodies (Fig. Sb, arrowhead) and stubs of the same diameter are visible on the E-face (Fig. 6b, arrowhead). As seen in Fig. 6, the lipid body plaques found on the E-faces of the protein bodies


491

FIG. 4. Lipid bodies (LB) are connected to the ER by narrow necks (arrow) approximately 30 nm in diameter. In this micrograph, it is clear that the lipid body monolayer (arrow) is directly continuous with the outer leaflet of the membrane bilayer of the ER. Untreated cell, 24 h (x I 10,000, bar = 0.2 Mm).

reveal two types of domains: smooth domains related to the

triacylglycerol core regions of the lipid bodies, and particle-rich surface domains (Fig. 6, a and c, arrows) corresponding to fracture faces of the monolayers surrounding the lipid bodies. Thus, the lipid body monolayer, at least where it adjoins a protein body membrane, appears to contain many integral membrane proteins. DISCUSSION Control of Lipid Mobilization. Large amounts of hydrolytic enzymes are synthesized and secreted in aleurone cells after GA3 stimulation (7, 17). Because extensive membrane surfaces (ER) are required to support the synthesis of large quantities of secretory proteins, and large numbers of membrane vesicles are required to transport the newly synthesized proteins to the cell surface, the demand for membrane components is very high during this period. Many of the phospholipids in the membranes that are synthesized in aleurone cells in response to GA3 (22) appear to be derived from precursors released by triacylglycerol hydrolysis (35). Thus, rapid and efficient mobilization of the limited reserves of storage lipid is essential in order for the cells to respond properly to GA3. In order for the products of triacylglycerol hydrolysis to be used most efficiently, lipid degradation should be stimulated at the same time that hydrolase synthesis and secretion are activated. Since GA3 triggers the latter process, it may affect lipid mobilization as well. Stimulation of triacylglycerol hydrolysis by GA3 would lead to an increase in the pool of free fatty acids. The stimulation of phospholipid synthesis by GA3 which has previously been reported (1, 10, 21, 23) may, in fact, be due to a GA3-induced increase in the pool of phospholipid precursors derived from triacylglycerol. We investigated one aspect of lipid mobilization that might be controlled by GA3 in barley aleurone cells, namely the relationship between lipase and the triacylglycerol in the lipid bodies. The results of our experiments indicate that the distribution of lipase in aleurone cells is hormonally controlled. In particular, we found that: (a) GA3 induces a shift in the distribution of lipase activity from a 10,000g pellet to a lipid body fraction, (b) the

shift can be inhibited if ABA is added simultaneously, and (c) the shift takes place around 1 h after the cells have been exposed to GA3.

Association of Lipase with 10,000g Pellet. In aleurone cells that have not been exposed to GA3, lipase activity is associated with a component that sediments at 10,000g. Because the 10,000g pellet contains a mixture of organelles, this component cannot be positively identified at this point. However, only protein bodies fulfill the necessary requirements for such a component: protein bodies sediment at 10,000g, contain hydrolytic enzymes, and associate with lipid bodies in vivo. Lipid bodies also associate with ER in intact cells; however, during homogenization, ER cisternae usually fragment into small vesicles that cannot be pelleted at 10,000g. The specific activity of lipase in microsomal fractions prepared by centrifuging the supernatant fraction at 100,000g for 1 h is at least 2-fold lower than the specific activity in the 10,000g pellet (data not shown). Glyoxysomes pellet at 10,000g and contain hydrolytic enzymes, but the nonspecific acyl hydrolases they contain are generally inactive on triacylglycerols (19). Although glyoxysomes are found in contact with lipid bodies in other germinating seeds (16), they do not interact with lipid bodies in any direct way in aleurone cells. On the other hand, we can identify protein body membranes in the 10,000g pellet and have been able to demonstrate that protein bodies form direct connections with lipid bodies in intact aleurone cells. Protein bodies contain a variety of different hydrolases (31, 36) which function during germination, including acid lipase (20). Unfortunately, our attempts to isolate a fraction enriched in intact protein bodies from aleurone protoplasts have been unsuccessful, most likely due to the fact that the dense inclusion bodies ofthe protein bodies tend to rupture the limiting membrane during isolation. Changes in Lipase Distribution. The results of the experiments on the distribution of lipase activity are intriguing. Our data indicate that a shift in the distribution of lipase activity occurs and that additional lipase activity becomes associated with the lipid bodies after the cells are exposed to GA3. These changes are not due to a change in the behavior of lipid bodies on sucrose

492



I? .; pp

.

*

'.

.r.j'I..

4-.

44. .

FIG. 5. Freeze-fracture electron micrographs of the P-face of protein body membranes (PB-PF). Untreated cell, 24 h. a, Lipid bodies (LB) completely surround protein bodies (PB) and associate with their membranes at sites which are marked by smooth plaques (arrows, *). The outer leaflet (PF) of the protein body membrane has an extremely high density of intramembrane particles (x50,000, bar = 0.4 Mm). b, High magnification view of the protein body membrane shown in (a). No steps are seen in the regions where the fracture plane crosses (arrows) from the interior of the lipid bodies (*) to the particle-rich outer leaflet of the protein body membrane (PB-PF). Direct contact between the two organelles is confined to a small round pore region which is visible on the P-face as a small pit (arrowhead) approximately 30 nm in diameter (x 100,000, bar = 0.2 ,um).

\1~~~~~~~~~~~~-.~ ~ ~ ~~~~34,

2~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ -w '-Xe-,{

4

>

?

i, t _

., ;S: S l

FIG. 6. Freeze-fracture electron micrographs of the E-face of protein body membranes (PB-EF). Untreated cells, 7 to 8 h. a, Lipid bodies (LB) associate with protein body membranes (PB) at sites which are marked by plaques (*). In E-face views, many plaques with particle-rich domains are visible (arrows). The rough surfaces are produced when a fracture plane passes along the P-face of a lipid body monolayer that is full of integral membrane proteins (x80,000, bar = 0.2 MArm). b, High magnification view of a smooth plaque (*) on the E-face of a protein body membrane. No step is seen in the transition zone (arrow) between the particle-rich inner leaflet of the protein body membrane (PB-EF) and the smooth area (*) that results when triacylglycerol in the interior of the lipid body is fractured. The arrowhead marks a stub corresponding to the narrow neck-like region between a lipid body and the protein body membrane (x75,000, bar = 0.2 Am). c, High magnification view of rough plaques on the E-face of the protein body membrane (PB-EF). Two types of domains are visible: a smooth triacylglycerol core region (*) and a particle-rich surface layer (LBM) corresponding to the inner surface of the lipid body monolayer (x95,000, bar = 0.2 ,um).

494

FERNANDEZ AND STAEHELIN LB monolayer

fracture face of triglyceride core

fracture face of LB monoloyer

7~ ~ ~ ~ ~ ~ ~ ~ ~' PROTEIN BODY PB membrane

FIG. 7. Diagram illustrating the relationship between the structural features seen in freeze-fracture electron micrographs (Figs. 5 and 6) and the fracture planes that pass through the sites where lipid bodies are attached to protein body membranes. The P-face of the protein body membrane is seen if the fracture is viewed from the bottom (protein body matrix); the E-face of the protein body membrane is seen if the fracture is viewed from the top (cytoplasm).

gradients, i.e. lipid bodies do not appear to detach from protein body membranes and float up after GA3 treatment. We find that, in general, the yield of lipid bodies from GA3-induced layers is lower than the yield from the same number of uninduced layers. Changes in the amount of lipase activity associated with lipid bodies could occur by either of two mechanisms: (a) de novo synthesis of lipase molecules that subsequently become associated with lipid bodies or (b) transfer of lipase from protein bodies to lipid bodies. Our data does not allow us unambiguously to distinguish between these two alternatives. We favor the lipase transfer hypothesis because of the following points: (a) the total lipase activity is not dramatically different in preparations from induced and uninduced cells; (b) the specific activity of lipase in the pellet is lower in preparations from induced cells; (c) the shift in distribution could occur very rapidly, i.e. within the time course observed; and (d) a transfer mechanism would explain why lipid bodies and protein bodies are so tightly associated. In addition, Gregerson and Taiz (15) report that, when aleurone cells are treated with ABA or ABA + GA3, lipid bodies accumulate in the cortical cytoplasm adjacent to the plasma membrane, i.e. away from the protein bodies. Thus, the cytoplasmic distribution of lipid bodies in aleurone cells does appear to be hormonally controlled to some extent. On the other hand, GA3induced de novo synthesis has been documented for several different proteins in aleurone layers (17), although not for lipases. Furthermore, in corn scutellar tissue, which contains very substantial amounts of storage lipid, lipase appears to be synthesized de novo during the course of germination (33). However, if de novo synthesis of lipase does occur in barley aleurone cells, then that synthesis must be very tightly controlled and subtle changes in the level of total lipase activity mtpst be occurring. To be consistent with our data, Jipase synthesis would have to be confined to a very narrow window of time because the shift in the distribution of lipase activity is initiated around 1 h after the cells are exposed to GA3 and is complete 1 h later. Total lipase activity cannot be measured accurately enough to see subtle changes because aleurone cels contain a wide variety of hydrolases, including several different proteases, that are released to different degrees during homogenization of different samples. In preliminary experiments, RNA transcription inhibitors (cordycepin) and protein translation inhibitors (cycloheximide) do not affect the shift in distribution in a consistent manner (data not shown). ER-Lipid Body Associations. Our micrographs of ultrarapidly frozen cells demonstrate unambiguously that lipid bodies asso-


ciate with other organelles, specifically the ER and protein bodies, during germination. The nature of the association with the ER that we have observed, i.e. continuity between the monolayer of the lipid body and the outer leaflet of the ER bilayer, is predicted by, and consistent with, one of the current models of lipid body biogenesis (34). According to this model, storage lipids accumulate in the interior of ER membranes as small droplets that, at maturity, bud off from the ER into the cytoplasm, pulling along a monolayer of membrane phospholipids and selected membrane proteins. Triacylglycerol synthesis may continue at low levels during germination, or some lipid bodies may remain permanently attached to the ER after their synthesis during seed development. The ER-lipid body association may also be involved in phospholipid synthesis or play some other, as yet unidentified, role during germination. Lipid Body-Protein Body Associations. The association of lipid bodies with the protein body membrane is even more intriguing. Although many investigators have noted this association in aleurone cells (4, 20, 22, 35), its nature was unclear. Using freezefracture electron microscopy, we have demonstrated that the monolayer of the lipid body and the outer leaflet of the bilayer of the protein body are continuous. The membrane continuity is confined to a very small area. Triacylglycerols in the lipid bodies do not appear to spread between the two halves of the protein body bilayer. Instead, the attachment is restricted to a narrow neck-like structure which is the same diameter whether the lipid body is associated with the ER or with the protein body. Fusion of lipid bodies with other cellular membranes does not appear to occur indiscriminately: we have only observed connections to ER and protein body membranes in our ultrarapidly frozen freeze-fracture samples. Once a connection is established, its size appears to be physically restrained, possibly by the presence of structural proteins in the lipid body membrane (29) and/ or the high density of intramembrane particles in the bilayer. To the best of our knowledge, no function for the intimate association of lipid bodies and protein bodies has ever been proposed, although Wilkinson et al. (35) suggest that the lipid body-protein body complex may be involved in phospholipid synthesis in wheat aleurone cells. Since protein bodies in cereals can arise as dilations of ER cisternae (24, 28) as well as from condensing Golgi vesicles (24), and it has been postulated that lipid bodies are synthesized on the ER (34), it is possible that lipid body-protein body associations are remnants of lipid bodyER associations and have no function in germination. However, the strength and specificity of the protein body-lipid body association argue against that possibility. The lipid body-protein body complex is maintained even when aleurone cells are subjected to high centrifugal forces. Chen and Jones (5) have stratified aleurone cells by centrifuging layers at 105,000g for 90 min. Although most of the lipid bodies float to the top of the cell, a certain proportion of the lipid bodies remain tightly associated with the protein bodies. Working Model for Mechanism of Transfer. Protein bodies serve as storage structures for a variety of hydrolytic enzymes that function during germination (31, 36). If protein bodies also store lipases and these lipases are in the membranes of the protein bodies, then one possible function of a lipid body-protein body association might be to join the onzyme and its substrate. Because the monolayer of the lipid bodies is continuous with the outer leaflet of the protein body membrane, certain complexes of integral membrane proteins could be easily transferred between the two organelles. Our model (Pig. 8) for how this transfer could occur consists of three main steps: (a) lipid bodies become associated with the protein bodies, (b) the monolayers of lipid bodies fuse with the outer leaflets of the bilayers of protein bodies, and (c) membrane complexes diffuse from the protein bodies to the lipid bodies. The transfer would also be a fairly


good system in which to examine the specifics of lipase biochemistry because the levels of lipase are relatively low. The results of our experiments suggest that the interaction between lipase and triacylglycerol may be controlled by regulating the distribution of the enzyme between the protein body membrane (storage form) and surface layer of lipid bodies (active form). However, it is clear that the postulated mechanism of lipase transfer between these two compartments will have to be tested more rigorously in systems that are more accessible to detailed bio-

LIPID BODY

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chemical analysis. .

Acknowledgments-We would like to thank Dr. Anthony Huang for his advice

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on lipase assays, Lisa Maynard and Elizabeth Amatruda for their help during the course of this project, and Dr. Felix Mauch for helpful comments on the manu-

PROTEIN BODY

script.

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PC3wPROTEIN CARBOiYDIRATE

BODY

FIG. 8. Model illustrating the mechanism by which lipases associated with protein body membranes may be transferred to lipid bodies. Lateral diffusion of lipase complpxes from the bilayer of the protein body into the monolayer of the lipid body would expose the active sites of the lipases to their substrate, the triacylglycerol stored within the lipid bodies. Tr4nsmembrane proteins would remain in the protein body membrane.

selective process. Complexes of integral membrane proteins that have hydrophilic surfaces exposed to both the cytoplasm and the matrix of the protein body would remain in the protein body membrane. In contrast, complexes of integral membrane proteins that lack hydrophilic surfaces on the side of the bilayer facing the protein body matrix could diffuse freely from the membrane to the lipid body. Functional lipases in lipid bodies are likely to have the latter structure because they must have access both to water in the cytoplasm and to the hydrophobic triacylglycerol core of the lipid bodies. The specific effects of GA3 on the transfer process are unclear. Lipid bodies are closely associated with protein bodies in untreated as well as GA3-treated cells. GA3 may induce changes in the ionic environment or in the composition or organization of the protein body membrane that facilitate diffusion of lipase complexes into these lipid bodies. Future Directions. In conclusion, our results suggest that the relationship between lipase and lipid bodies is dynamic and may be subject to hormonal control. To characterize the functional significance of this relationship more thoroughly in aleurone cells, additional tools, particularly lipase-specific antibodies and cDNA probes for lipase messages, are needed. The barley aleurone system is important because it is presently the only system where hormonal control of processes that occur during germination can be clearly demonstrated. However, it may not be a

LITERATURE ClTlE D 1. BEN-TAL Y, JE VARNER 1974 An early response to gibberellic acid not requiring protein synthesis. Plant Physiol 54: 813-816 2. BRADFORD MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizng the principle of protein-dye binding. Anal Biochem 72: 248-254 3. BRANTON DS, S BULLIVANT, NG GILULA, MJ KARNOVSKY, H Mooit, K MOHLETHALER, D NORTHCOTE, L PACKIER, P SATIR, V SPETH, LA STAEHELIN, RL STEERE, RS WEINSTEIN 1975 Frez-etching nomenclature. Science 190: 54-56 4. BU RrOSE M 1971 Ultrastructure of barley aleurone cells as shown by freezeetching. Planta 96: 13-26 5. CHEN R, RL JONES 1974 Studies on the release of barley aleurone cell proteins: autoradiography. Planta 119: 207-220 6. CHRISPEELS, MJ, JE VARNER 1966 Inhibition of gibberellic acid induced formation of a-amylase by abscisin II. Nature 212: 1066-1067 7. CHRISPEELS, MJ, JE VARNER 1967 Gibberellic acid-enhanced synthesis and release of a-amylase and ribonuclease by isolated barley aleurone layers. Plant Physiol 42: 397-406 8. CLARKE NA, MC WILKINSON, DL LAIDMAN 1983 Lipid metabolism in germinating cereals. In PJ Barnes, ed, Lipids in Cereal Technology. Academic Press, London, pp 57-92 9. DoIG RI, AJ COLBORNE, G MORRIS, DL LAIDMAN 1975 The induction of glyoxysomal enzyme activities in the aleurone cells of germinating wheat. J Exp Bot 26: 387-398 10. EVINS WH, JE VARNER 1971 Hormone-controlled synthesis of endoplasmic reticulum in barley aleurone cells. Proc Natl Acad Sci USA 68: 1631-1633 1 1. EVINs WH, JE VARNER 1972 Hormonal control of polyribosome formation in barley aleurone layes Plant Physiol 49: 348-352 12. FERNANDEZ DE, LA STAEHELIN 1985 Structural organization of ultrarapidlyfrozen barley aleurone cells actively involved in protein secretion. Planta 165: 455-468 13. FERNANDEZ DE, LA STAEHELIN 1987 Effect of gibberellic acid on lipid degradation in barley aleurone layers In JE Fox, M Jacobs, eds, Molecular Biology of Plant Growth Control, UCLA Symposia on Molecular and Cellular Biology, New Series, Vol 44. Alan R Liss, Inc, New York, pp 323-334 14. FIRN RD, H KENDE 1974 Some effects of applied gibberellic acid on the synthesis and degradation of lipids in isolated barley aleurone layers. Plant Physiol 54: 911-915 15. GREGERSON EL, L TAIZ 1985 The effect of abscisic acid on the ultrastructure of barley aleurone cells. Bot Gaz 146: 1-5 16. GRUBER PJ, RN TRELEASE, WM BECKER, EH NEWCOMB 1970 A correlative 17.

18. 19. 20.

21. 22.

23.

ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds. Planta 93: 269-288 HIGGINS TJV, JV JACOBSEN, JA ZWAR 1982 Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers. Plant Mol Biol 1: 191-215 HOOLEY R 1982 Protoplasts isolated from aleurone layers of wild oat (Avena fatua L.) exhibit the classic response to gibberellic acid. Planta 154: 29-40 HUANG AHC 1984 Plant lipases. In HL Brockman, B Borgstrom, eds, Lipases. Elsevier Press, Amsterdam, pp 419-442 JELSEMA CL, DJ MORRt, M RUDDAT, C TURNER 1977 Isolation and characterization of the lipid reserve bodies, spherosomes, from aleurone layers of wheat. Bot Gaz 138: 138-149 JOHNSON KD, H KENDE 1971 Hormonal control oflecithin synthesis in barley aleurone cells: regulation of the CDP-choline pathway by gibberellin. Proc Natl Acad Sci USA 68: 2674-2677 JONES RL 1969 Gibberellic acid and the fine structure of barley aleurone cells: I. Changes during the lag phase of a-amylase synthesis. Planta 87: 119-133 KOEHLER DE, JE VARNER 1973 Hormonal control of orthophosphate incorporation into phospholipids of bariey aleurone layers. Plant Physiol 52: 208214

24. KRISHNAN HB, VR FRANCESCHI, TW OKrrA 1986 Immunochemical studies on the role of the Golgi complex in protein-body formation in rice seeds.

Planta 169: 471-480

496


25. LIN Y-H, AHC HUANG 1984 Purification and initial characterization of lipase from the scutella of corn seedlings. Plant Physiol 76: 719-722 26. MORRISON WR 1983 Acyl lipids in cereals. In PJ Barnes, ed, Lipids in Cereal Technology. Academic Press, London, pp 11-32 27. NIXON M, SHP CHEN 1979 A simple and sensitive colorimetric method for the detennination of long-chain free fatty acids in subcellular organelles. Anal Biochem 97: 403409 28. OPARKA KJ, N HARRIS 1982 Rice protein-body formation: all types are initiated by dilation of the endoplasmic reticulum. Planta 154: 184-188 29. Qu R, S-M WANG, Y-H LIN, VB VANCE, AHC HUANG (1986) Characteristics and biosynthesis of membrane proteins of lipid bodies in the scutella of maize (Zea mays L.). Biochem J 235: 57-65 30. TAVENER RJA, DL LAIDMAN 1972 The induction of lipase activity in the germinating wheat grain. Phytochemistry 11: 989-997


31. VAN DER WILDEN W, EM HERMAN, MJ CHRISPEELS 1980 Protein bodies of mung bean cotyledons as autophagic organelles. Proc Natl Acad Sci USA 77: 428-432 32. VARTY K, DL LAIDMAN 1976 The pattern and control of phospholipid metabolism in wheat aleurone tissue. J Exp Bot 27: 748-758 33. WANG S-M, AHC HUANG 1987 Biosynthesis of lipase in the scutellum of maize kernel. J Biol Chem 262: 2270-2274 34. WANNER G, H FORMANEK, RR THEIMER 1981 The ontogeny of lipid bodies (spherosomes) in plant cells: Ultrastructural evidence. Planta 151: 109-123 35. WILKINSON MC, DL LAIDMAN, T GALLIARD 1984 Two sites of phosphatidylcholine synthesis in the wheat aleurone cell. Plant Sci Lett 35: 195-199 36. YATSU LY, TJ JACKS 1968 Association of lysosomal activity with aleurone grains in plant seeds. Arch Biochem Biophys 124: 466-471 37. YATSU LY, TJ JACKS 1972 Spherosome membranes: half unit-membranes. Plant Physiol 49: 937-943